Mid-infrared (IR) spectroscopic imaging using inherent vibrational contrast has been broadly used as a powerful analytical tool for sample identification and characterization. However, the low spatial resolution and large water absorption associated with the long IR wavelengths hinder its applications to study subcellular features in living systems. Recently developed mid-infrared photothermal (MIP) microscopy overcomes these limitations by probing the IR absorption–induced photothermal effect using a visible light. MIP microscopy yields submicrometer spatial resolution with high spectral fidelity and reduced water background. In this review, we categorize different photothermal contrast mechanisms and discuss instrumentations for scanning and widefield MIP microscope configurations. We highlight a broad range of applications from life science to materials. We further provide future perspective and potential venues in MIP microscopy field.

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

Midinfrared photothermal (MIP) microscopy, also called optical photothermal infrared (O-PTIR) microscopy, is an emerging tool for bond-selective chemical imaging of living biological and material samples. In MIP microscopy, a visible probe beam detects the photothermal-based contrast induced by a vibrational absorption. With submicron spatial resolution, high spectral fidelity, and reduced water absorption background, MIP microscopy has overcome the limitations in infrared chemical imaging methods. In this review, we summarize the basic principle of MIP microscopy, the different origins of MIP contrasts, and recent technology development that pushed the resolution, speed, and sensitivity of MIP imaging to a new stage. We further emphasize its broad applications in life science and material characterization, and provide a perspective of future technical advances.

By optically sensing absorption-induced photothermal effect, mid-infrared (IR) photothermal (MIP) microscope enables super-resolution IR imaging of biological systems in water. However, the speed of current sample-scanning MIP system is limited to milliseconds per pixel, which is insufficient for capturing living dynamics. By detecting the transient photothermal signal induced by a single IR pulse through fast digitization, we report a laser-scanning MIP microscope that increases the imaging speed by three orders of magnitude. To realize single-pulse photothermal detection, we use synchronized galvo scanning of both mid-IR and probe beams to achieve an imaging line rate of more than 2 kilohertz. With video-rate speed, we observed the dynamics of various biomolecules in living organisms at multiple scales. Furthermore, by using hyperspectral imaging, we chemically dissected the layered ultrastructure of fungal cell wall. Last, with a uniform field of view more than 200 by 200 square micrometer, we mapped fat storage in free-moving Caenorhabditis elegans and live embryos.

By optically sensing the mid-infrared absorption induced photothermal effect, midinfrared photothermal (MIP) microscope enables super-resolution IR imaging and scrutinizing of biological systems in an aqueous environment. However, the speed of current lock-in based sample-scanning MIP system is limited to 1.0 millisecond or longer per pixel, which is insufficient for capturing dynamics inside living systems. Here, we report a single pulse laserscanning MIP microscope that dramatically increases the imaging speed by three orders of magnitude. We harness a lock-in free demodulation scheme which uses high-speed digitization to resolve single IR pulse induced contrast at nanosecond time scale. To realize single pulse photothermal detection at each pixel, we employ two sets of galvo mirrors for synchronized scanning of mid-infrared and probe beams to achieve an imaging line rate over 2 kHz. With video-rate imaging capability, we observed two types of distinct dynamics of lipids in living cells. Furthermore, by hyperspectral imaging, we chemically dissected a single cell wall at nanometer scale. Finally, with a uniform field of view over 200 by 200 μm2and 2 Hz frame rate, we mapped fat storage in free-movingC. elegansand live embryos.